Leishmania major, serine/threonine protein phosphatase, putative
Progress of Research:
LmajSTPP gene successfully constructed through Primary PCR and amplified by Secondary PCR.
Figure 1. 1% agarose gel of primary PCR product of L. maj STPP gene and secondary PCR product with 1st and last oligo primers. Lane 1 contained the primary PCR product and lane 2 contained the secondary PCR product.
Figure 2. 1% agarose gel secondary PCR products of L.majSTPP gene using tail primers. Lane 1 was for another study. Lane 2 is the secondary PCR product of LmajSTPP gene.
Background and Introduction:
*Target (protein/gene name):serine/threonine protein phosphatase *NCBI Gene # or RefSeq#:5652543 (?) *Protein ID (NP or XP #) or Wolbachia#: ?? (used Daniel's Oligo Design - Dr. B090313)
*Organism (including strain):Leishmania major *Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents *Enzyme Assay information: Assay for Calcineurin (3.1.3.16 ) ?? SIgma - just use PNPP via BRENDA *Druggability: 0.8 *Essentiality Data –yes *Abundant Essentiality Data on TDR page* http://tdrtargets.org/targets/view?gene_id=29295
Background/Disease: Leishmaniasis is a disease caused by protozoan parasites that belong to the genus of Leishmania and is transmitted by the bit of certain species of sand fly. Leishmaniasis threatens about 350 million men, women and children in 88 countries around the world. As many as 12 million people are believed to be currently infected, with about 1–2 million estimated new cases occurring every year. The disease can have a wide range of clinical symptoms, which may be cutaneous,mucocutaneous or visceral. Cutaneous leishmaniasis is the most common form. Visceral leishmaniasis is the most severe form, in which vital organs of the body are affected.
(Extracellular dephosphorylation in the parasite, Leishmania major. This protein “may play a significant role in host cell-parasite recognition and infection.”)
Current Treatments: There are two common therapies containing antimony (known as pentavalent antimonials): meglumine antimoniate (Glucantime) and sodium stibogluconate (Pentostam). It is not completely understood how these drugs act against the parasite; they may disrupt its energy production ortrypanothione metabolism. Unfortunately, in many parts of the world, the parasite has become resistant to antimony when treating for visceral or mucocutaneous leishmaniasis,[5] but the level of resistance varies according to species
Miltefosine (Impavido), is a new drug for visceral and cutaneous leishmaniasis. The cure rate of miltefosine in phase III clinical trials is 95%; studies in Ethiopia show it is also effective in Africa. However, there are problems associated with the use of miltefosine that arise from its teratogenicity and pharmacokinetics: In a Dutch study by Thomas P.C. Dorlo in 2008, miltefosine was shown to be much slower eliminated from the body than previously thought and was therefore still detectable in human plasma samples taken five months after the end of treatment. The presence of subtherapeutic miltefosine concentrations in the blood beyond five months after treatment might contribute to the selection of resistant parasites and, moreover, the measures for preventing the teratogenic risks of miltefosine must be reconsidered. This led to some reluctance to taking Miltefosine by affected populations.
Sequence of the gene produced from primer overlap:
ATGACTACCGCTGGTGGTGGTTCTGCGGTGGGTTCTTCTTCCGCTCTCGACCTCGACGAA ATGATCAACTACGTTATCCAGTGCAAACCGCTGTCTGAACAGCAGGTTGCGCGCCTGTGC GAAAAAGTCAAAGAAGTTCTGGAGAAAGAAAACAACGTTCATGCGGTTCGTGCGCCGGTT ACCGTTTGCGGTGACGTTCACGGTCAATTCCACGACCTCCTGGAACTCTTCAAAATCGGT GGTCTGCCTCCGGACACCAATTACCTGTTCATGGGTGACTACGTCGATCGCGGTTATTAC AGCGTTGAAACGGTGACCCTCCTGCTGCTGTACAAACTGCGTTACCCGCAGCGTCTGCAC CTCCTCCGTGGTAACCACGAGTCTCGCCAGATTACTCAGGTTTACGGTTTCTACGACGAA TGCATCCGCAAATACGGCTCTGCGAATGTTTGGACCATCTTCACCGACCTCTTCGACTAC CTCCCGCTGACCGCGCTGGTTGAAAACGACATCTTCTGCCTGCACGGCGGTCTGTCTCCG ACCGTTGACACCTTCTCTCACATCCGTAACCTGGACCGTGTTCAGGAAGTTCCGCACGAG GGTCCGATGTGCGATCTGCTCTGGAGCGATCCGGACGACCGCGACGGTTGGGGTATCTCT CCGCGTGGTGCGGGTTTTACGTTCGGTCAGGGTGTAACCGAGGGTTTCTGCCACAACAAC AAAATCAAAACCATCGCGCGTGCGCATCAGCTGGTTATGGACGGTTACTCTTGGACCCAC CAAGATCAGCTCGTTACCATTTTCTCTGCGCCGAACTACTGCTACCGTTGCGGTAATCTC GCGGGTCTGCTGGAGCTGGACGAACACATGAATAAATGCTTTTTCCAATTTGACCCGGCG TAA
Primers for pNIC-Bsa4 cloning: Forward primers: TACTTCCAATCCATGACTACCGCTGGTGGTGG Reverse primers: TATCCACCTTTACTGTTACGCCGGGTCAAATTGG
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
Daniel's:
Progress of Research:
LmajSTPP gene successfully constructed through Primary PCR and amplified by Secondary PCR.Background and Introduction:
*Target (protein/gene name): serine/threonine protein phosphatase
*NCBI Gene # or RefSeq#: 5652543 (?)
*Protein ID (NP or XP #) or Wolbachia#: ?? (used Daniel's Oligo Design - Dr. B090313)
*Organism (including strain): Leishmania major
*Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents
*Enzyme Assay information: Assay for Calcineurin (3.1.3.16 ) ?? SIgma - just use PNPP via BRENDA
*Druggability: 0.8
*Essentiality Data –yes
*Abundant Essentiality Data on TDR page*
http://tdrtargets.org/targets/view?gene_id=29295
http://tritrypdb.org/tritrypdb/showRecord.do?name=GeneRecordClasses.GeneRecordClass&primary_key=LmjF25.1320
EC#:3.1.3.16
http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.16
PDB # or closest PDB entry is using homology model: 3ICF (homology model- 94% query coverage)
Comparison Protein BLAST Results:
---- Query Coverage: 94%
---- Max % Identities: 39%
---- % Positives 56%
---- Chain used for homology: To be determined
>3ICF:A|PDBID|CHAIN|SEQUENCE
KNAFKGAKIKNMSQEFISKMVNDLFLKGKYLPKKYVAAIISHADTLFRQEPSMVELENNSTPDVKISVCGDTHGQF
YDVLNLFRKFGKVGPKHTYLFNGDFVDRGSWSCEVALLFYCLKILHPNNFFLNRGNHESDNMNKIYGFEDECKYK
YSQRIFNMFAQSFESLPLATLINNDYLVMHGGLPSDPSATLSDFKNIDRFAQPPRDGAFMELLWADPQEANGMGP
SQRGLGHAFGPDITDRFLRNNKLRKIFRSHELRMGGVQFEQKGKLMTVFSAPNYCDSQGNLGGVIHVVPGHGILQ
AGRNDDQNLIIETFEAVEHPDIKPMAYSNGGFGL
Background/Disease: Leishmaniasis is a disease caused by protozoan parasites that belong to the genus of Leishmania and is transmitted by the bit of certain species of sand fly. Leishmaniasis threatens about 350 million men, women and children in 88 countries around the world. As many as 12 million people are believed to be currently infected, with about 1–2 million estimated new cases occurring every year. The disease can have a wide range of clinical symptoms, which may be cutaneous,mucocutaneous or visceral. Cutaneous leishmaniasis is the most common form. Visceral leishmaniasis is the most severe form, in which vital organs of the body are affected.
(Extracellular dephosphorylation in the parasite, Leishmania major. This protein “may play a significant role in host cell-parasite recognition and infection.”)
Current Treatments:
There are two common therapies containing antimony (known as pentavalent antimonials): meglumine antimoniate (Glucantime) and sodium stibogluconate (Pentostam). It is not completely understood how these drugs act against the parasite; they may disrupt its energy production ortrypanothione metabolism. Unfortunately, in many parts of the world, the parasite has become resistant to antimony when treating for visceral or mucocutaneous leishmaniasis,[5] but the level of resistance varies according to species
Miltefosine (Impavido), is a new drug for visceral and cutaneous leishmaniasis. The cure rate of miltefosine in phase III clinical trials is 95%; studies in Ethiopia show it is also effective in Africa. However, there are problems associated with the use of miltefosine that arise from its teratogenicity and pharmacokinetics: In a Dutch study by Thomas P.C. Dorlo in 2008, miltefosine was shown to be much slower eliminated from the body than previously thought and was therefore still detectable in human plasma samples taken five months after the end of treatment. The presence of subtherapeutic miltefosine concentrations in the blood beyond five months after treatment might contribute to the selection of resistant parasites and, moreover, the measures for preventing the teratogenic risks of miltefosine must be reconsidered. This led to some reluctance to taking Miltefosine by affected populations.
Image of protein (PyMol or etc):
PDB code: 3ICF
CDS:
ATGACGACGGCCGGCGGCGGATCGGCGGTGGGGTCCAGCAGCGCCCTCGACTTGGATGAG
ATGATTAATTACGTCATACAGTGCAAGCCGCTCAGCGAACAGCAGGTCGCGCGCCTGTGC
GAGAAGGTGAAGGAGGTGCTGGAGAAGGAGAACAACGTGCACGCGGTGAGGGCGCCAGTC
ACGGTGTGCGGCGATGTGCATGGCCAGTTTCACGACTTGCTGGAGCTTTTCAAGATTGGC
GGGTTGCCGCCGGACACGAACTACCTGTTCATGGGGGACTACGTGGACCGCGGCTACTAC
AGCGTCGAGACGGTGACGCTGCTCCTCCTCTACAAGCTGCGATACCCCCAGCGACTTCAT
CTTCTCCGCGGCAACCACGAGTCGCGCCAGATCACGCAGGTGTATGGCTTCTACGATGAG
TGCATTCGCAAGTATGGTAGCGCCAACGTCTGGACGATCTTCACGGACCTGTTTGACTAC
CTGCCCCTGACAGCGTTGGTCGAGAACGACATCTTCTGCCTTCACGGTGGTCTCTCACCG
ACGGTCGACACATTCAGCCACATCCGCAACCTCGACCGCGTGCAGGAAGTGCCGCACGAG
GGGCCGATGTGCGATCTGCTATGGTCGGACCCGGACGATCGTGATGGCTGGGGCATCAGC
CCCCGTGGCGCCGGCTTCACCTTCGGCCAAGGAGTCACCGAAGGCTTCTGCCACAACAAC
AAGATCAAGACAATCGCCCGTGCGCATCAGCTTGTCATGGACGGCTACAGCTGGACGCAT
CAGGATCAGCTCGTCACGATCTTCAGCGCACCAAACTACTGCTACCGCTGCGGCAACCTC
GCCGGTCTTTTGGAGCTTGATGAGCACATGAACAAGTGCTTCTTTCAGTTCGACCCGGCG
CCGCGACGCGGTGAAGCGCAGGTGTCGAAGAAGACGCCGGACTATTTTCTATAG
Amino Acid Sequence:
XP_001683887.1MTTAGGGSAVGSSSALDLDEMINYVIQCKPLSEQQVARLCEKVKEVLEKENNVHAVRAPV
TVCGDVHGQFHDLLELFKIGGLPPDTNYLFMGDYVDRGYYSVETVTLLLLYKLRYPQRLH
LLRGNHESRQITQVYGFYDECIRKYGSANVWTIFTDLFDYLPLTALVENDIFCLHGGLSP
TVDTFSHIRNLDRVQEVPHEGPMCDLLWSDPDDRDGWGISPRGAGFTFGQGVTEGFCHNN
KIKTIARAHQLVMDGYSWTHQDQLVTIFSAPNYCYRCGNLAGLLELDEHMNKCFFQFDPA
PRRGEAQVSKKTPDYFL
length of your protein in Amino Acids:
317 aaMolecular Weight of your protein
35861 DaSequence of the gene produced from primer overlap:
ATGACTACCGCTGGTGGTGGTTCTGCGGTGGGTTCTTCTTCCGCTCTCGACCTCGACGAA ATGATCAACTACGTTATCCAGTGCAAACCGCTGTCTGAACAGCAGGTTGCGCGCCTGTGC GAAAAAGTCAAAGAAGTTCTGGAGAAAGAAAACAACGTTCATGCGGTTCGTGCGCCGGTT ACCGTTTGCGGTGACGTTCACGGTCAATTCCACGACCTCCTGGAACTCTTCAAAATCGGT GGTCTGCCTCCGGACACCAATTACCTGTTCATGGGTGACTACGTCGATCGCGGTTATTAC AGCGTTGAAACGGTGACCCTCCTGCTGCTGTACAAACTGCGTTACCCGCAGCGTCTGCAC CTCCTCCGTGGTAACCACGAGTCTCGCCAGATTACTCAGGTTTACGGTTTCTACGACGAA TGCATCCGCAAATACGGCTCTGCGAATGTTTGGACCATCTTCACCGACCTCTTCGACTAC CTCCCGCTGACCGCGCTGGTTGAAAACGACATCTTCTGCCTGCACGGCGGTCTGTCTCCG ACCGTTGACACCTTCTCTCACATCCGTAACCTGGACCGTGTTCAGGAAGTTCCGCACGAG GGTCCGATGTGCGATCTGCTCTGGAGCGATCCGGACGACCGCGACGGTTGGGGTATCTCT CCGCGTGGTGCGGGTTTTACGTTCGGTCAGGGTGTAACCGAGGGTTTCTGCCACAACAAC AAAATCAAAACCATCGCGCGTGCGCATCAGCTGGTTATGGACGGTTACTCTTGGACCCAC CAAGATCAGCTCGTTACCATTTTCTCTGCGCCGAACTACTGCTACCGTTGCGGTAATCTC GCGGGTCTGCTGGAGCTGGACGAACACATGAATAAATGCTTTTTCCAATTTGACCCGGCG TAA
Primers for pNIC-Bsa4 cloning:
Forward primers: TACTTCCAATCCATGACTACCGCTGGTGGTGG
Reverse primers: TATCCACCTTTACTGTTACGCCGGGTCAAATTGG
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
Daniel's:
Fin's: https://drive.google.com/?authuser=1#folders/0B4O2KqKh2q_-aTdxaGQzMWVUbXc
Manuel's:
References:
Homology Model Info
SCRWL server
http://www.who.int/leishmaniasis/en/
http://www.cdc.gov/parasites/leishmaniasis/treatment.html